As normal cells progress towards tumorigenicity they must switch to an angiogenic phenotype in order to attract the nourishing new blood vessels on which their progressive growth depends. In human cells of glial or fibroblast origin this essential shift to an angiogenic phenotype requires loss of a tumor suppressor gene and results from a decrease in the secretion of an inhibitor of angiogenesis, thrombospondin-1 (TSP-1). Small peptides derived from the large TSP-l molecule are also potent inhibitors of neovascularization. This proposal outlines experiments designed to (l) test the in vivo efficacy of TSP-1 by examining its ability to slow tumor growth and angiogenesis when over and under expressed in human glioblastoma lines and by testing the effect of high levels of circulating TSP-1 on the growth of lung metastases; (2) use substituted peptides to define the specific amino acid residues that are necessary to the inhibitory activity of TSP-l peptides; (3) examine hallmarks of endothelial cell activation at the cell surface and in the nucleus to determine if TSP-1 and its peptides make endothelial cells unable to respond to angiogenic factors by blocking the propagation of positive signals; and (4) isolate another naturally occurring protein that inhibits angiogenesis and whose production depends on the retinoblastoma tumor suppressor gene. It is our hope that these experiments will increase our understanding of endothelial cell activation and its inhibition, prepare TSP-1 and its peptides for development as clinical anti-tumor agents and identify additional antiangiogenic proteins that will shed light on the function of tumor suppressor genes and may be of eventual clinical utility.